Shellstock refrigeration after harvesting is recommended to prevent further increases in Vibrio vulnificus numbers in oysters, but it could potentially induce a cold shock response in this bacterium. V. vulnificus was incubated at 35, 25, 20, and 15 degrees C and then subjected to 7.2 and 4 degrees C for 1 week. A cold-adaptation response that enhanced cell culturability was observed when cells were incubated at 15 degrees C prior to cold shock at 7.2 degrees C. In vitro cold shock gene expression was analyzed by reverse transcriptase PCR (RT-PCR). The expression of cold shock genes csp1 and csp5 (homologous genes to cspA and cspV) remained constant, despite cold shock. However, the transcript of csp3 was constitutively expressed before and after cold shock, with a few exceptions. The synthesis of csp3 mRNA in V. vulnificus C7184Tr (an avirulent strain) was induced only after 15 degrees C incubation and cold shock at 4 degrees C. The expression of csp4 was repressed after cold shock. Our data showed that the csp(s) tested in this study are not cold inducible. The transcripts of two oxidative stress-related genes, oxyR and katG, showed different induction patterns among strains after cold shock, suggesting that V. vulnificus cells encountered oxidative stress during cold shock.

Aims:  To determine Vibrio vulnificus response to shellstock refrigeration conditions while the bacterium was embedded in oysters (in vivo). Methods and Results:  Depurated oysters were artificially inoculated with V. vulnificus. Several cold‐shock conditions were examined according to the National Sanitation Shellfish Program guidelines. Culturability of cells along the refrigeration period was measured using specific colony dot‐blot. Gene expression of putative cold‐shock genes (csp1, csp3, csp4 and csp5) as well as three stress‐related genes (rpoS, oxyR and katG) was determined by reverse transcriptase polymerase chain reaction. Vibrio vulnificus exhibited a decline in cell viability after cold shock but a cold adaptation response was observed when oysters were kept at suboptimal (15°C) temperatures. 16SrRNA, and rpoS genes were constitutively expressed while expression of csp genes varied among strains and time points. Conclusions:  Vibrio vulnificus culturability was reduced after oysters were subjected to shellstock refrigeration conditions. When V. vulnificus was allowed to acclimate to cold temperatures, its survival after cold shock was higher. None of the cold shock genes analysed behaved as csp type I genes. Significance and Impact of the Study:  A model for artificially inoculated specific strains of V. vulnificus into oysters has been established. For the first time, V. vulnficus gene expression was assessed with the pathogen embedded in oysters.